The mechanism by which Lumazine Protein functions in the bioluminescence reaction with bacterial luciferase will be studied using fluorescence spectra, lifetime and anisotropy decay techniques. The natural ligand, 6,7-dimethyl-8-ribityllumazine can be replaced with analogs to determine the factors responsible for binding and function of this protein. Major use will be made of a Picosecond Fluorescence Lifetime system for this research. This is an NIH-University facility and uses a sync-pumped dye laser as and excitation source. Modifications of the protein structure will also be introduced to see what effects these have on both fluorescence and bioluminescence spectral properties. Modifications will be by using natural variants of lumazine protein whose primary sequence will be needed to be determined, by derivitizing certain amino acid residues, and by site-specific mutation, if the lumazine protein is successfully cloned. This cloning project is already underway in collaboration with another group. The effect of these modifications on certain of the protein properties will also be studied. Crystallization of lumazine proteins and their complexes with luciferases will also be done. This will enable some preliminary 3-dimensional structural information to be measured and collaboration with a fully equipped X-ray structure group then to be developed.